This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The research focus in the Malkowski laboratory is to understand how different classes of integral membrane enzymes utilize stereoselective oxygenations to generate unique oxylipins from a defined set of polyunsaturated fatty acid (PUFA) substrates. Oxylipins are bioactive lipid mediators found in plants and animals that are biosynthesized from 18-22 carbon PUFAs through the addition of one, two, or four molecules of oxygen via the catalytic activity of cytochrome P450s, lipoxygenases (LOX), and cyclooxygenases (COX), respectively. Diffraction studies are focused on three different integral membrane enzymes involved in oxylipin production in plants, animals, and invertebrates: a) pathogen-inducible oxygenase (PIOX) from Oryza sativa;b) human and murine cyclooxygenase-2 (COX-2);and c) a cyclooxygenase enzyme from the Caribbean coral Plexaura homomalla. Initial diffraction experiments of COX-2, carried out at the National Synchrotron Light Source (beamline X4A;March 2005), confirm that the crystals are in fact protein;however, diffraction was only observed to 6.5[unreadable] resolution. This project requires synchrotron radiation for two major reasons. First, while crystals of COX-2 grow large, they diffract only weakly to 8-10 [unreadable] on laboratory X-ray sources. Second, their relatively empty unit cell, combined with their sensitivity to any change in the detergent environment, make handling these crystals for data collection difficult. Often many crystals must be screened to find one that maintains minimal mosaicity (<2[unreadable]a) and reasonable diffraction.